Abstract The angiopoietins are a small class of secreted glycoproteins that play key roles in the maturation and maintenance of the mammalian vascular and lymphatic systems. They exert their effects through the Tie receptor tyrosine kinases. All four angiopoietins (Ang1-4) directly bind Tie2, while none of them binds the related Tie1, although the latter has been shown to modulate the Ang/Tie2 signaling. Integrins, and in particular a5b1, are involved in regulating Ang1-dependent angiogenesis and were shown to directly interact with Tie2. The Ang/Tie signaling is unique among receptor kinase-ligand systems in that distinct angiopoietin ligands, although all highly homologous, may function as agonist or antagonist in a context dependent manner. A detailed understanding of the mechanisms of Angiopoietin/Tie signaling and their precise function during angiogenesis requires a comprehensive structural and biophysical analysis of the angiopoietins, of the Tie receptors and their co-receptors, as well as of their interactions. We will use X-ray crystallography, combined with other biophysical, biochemical and cell-biological techniques, to study these molecules. We have previously determined the structures of Tie2, Ang1, Ang2, as well as of the Tie2/Ang1 and Tie2/Ang2 complexes, revealing important and unique characteristics of Tie2 signaling initiation. We also performed FRET-based studies on live cells to monitor the Tie1/Tie2 interactions on the cell surface. An important conclusion of our experiments is that the distinct signaling properties of the individual angiopoietins are likely the result of Tie2 co-receptors that respond differently to the different ligands. Therefore, we now propose to study the molecular mechanisms by which co-receptors modulate, in a regulated and cell/context-dependent manner, the Ang/Tie2 signaling events. We specifically propose to investigate the roles of Tie1 and intergrin a5b1. Finally, our ultimate goal is to determine crystal structures of the complete Tie2 receptor, including its transmembrane region, alone and in complex with ligands and co- receptors, and we will work on the production and structural characterization of functional full-length Tie proteins. + PHS 398/2590 (Rev. 05/01)